ANTICANCER RESEARCH 27: 2269-2274 (2007)

Frequent Promoter Methylation of M- in Hepatocellular Carcinoma is Associated with Poor Prognosis

SUGURU YAMADA, SHUJI NOMOTO, TSUTOMU FUJII, SHIN TAKEDA, NAOHITO KANAZUMI, HIROYUKI SUGIMOTO and AKIMASA NAKAO

Department of Surgery II, Graduate School and Faculty of Medicine, University of Nagoya, Nagoya, Japan

Abstract. Background: Many (CDH) are associated Transcriptional silencing of tumor suppressor and with various types of cancer and their genetic and epigenetic mismatch repair due to hypermethylation of 5’CpG alterations might be involved in carcinogenesis. Materials and islands is observed in a variety of malignancies (3, 4). Methods: We examined the methylation status of CDH genes in Several reports indicated that tumorigenesis is related to primary hepatocellular carcinoma (HCC) and corresponding loss of function of a tumor suppressor as a result of noncancerous liver tissues derived from 47 patients, and loss of heterozygosity (LOH), gene mutations, and evaluated the correlation with clinicopathological parameters. hypermethylation of promoter regions (5). Notably, allelic Results: Hypermethylation was detected at a ratio ranging from imbalance at 17p13 and frequent mutations of 0% to 55.3%. In particular, M-cadherin (CDH15) was the the p53 gene have been correlated with exposure to most hypermethylated of 7 CDH genes. Patients with aflatoxin B1 and chronic HBV infection (6). Mutations of methylated M-cadherin had shorter 5-year survival rates than ‚-catenin or its binding partner axin 1, leading to abnormal patients with unmethylated M-cadherin (overall survival rates, activation of the Wnt pathway, have been implicated in 67.4% vs. 82.7%; p=0.0167) when assessed using Kaplan- about one third of HCC cases (7, 8). Meier curves. Multivariate analysis revealed that M-cadherin In recent years, there has been increasing interest in a methylation status was an independent predictor of survival. superfamily of transmembrane , the cadherins Conclusion: We found that M-cadherin methylation status has (CDH). Cadherins are prime mediators of calcium-dependent prognostic significance for the poorer survival of patients with cell- in normal cells and are also involved in HCC. This is the first definitive report of a correlation between contact inhibition of cell growth by inducing cell cycle arrest M-cadherin and the prognosis of patients with HCC. (9, 10). Many cadherins are associated with cancer. For example, the epithelial E-cadherin (CDH1), a putative tumor Hepatocellular carcinoma (HCC) is one of the most suppressor, is deregulated in HCC, esophageal, gastric, colon, common malignancies worldwide and is generally associated pancreatic, lung, breast and prostate cancer (11-17). In with a poor prognosis (1). The disease is highly prevalent in contrast, increased expression of other cadherins such as N- Asia but relatively rare in developed countries, although its cadherin (CDH2), P-cadherin (CDH3), and OB-cadherin incidence is increasing in both the United States and Japan (CDH11) are clinically correlated with breast cancer (18, 19). (2). In many cases, HCC develops from chronic liver disease Aberrant methylation of H-cadherin (CDH13) associated with due to infection with hepatitis B virus (HBV), or hepatitis C gene silencing has been reported in several primary tumors virus (HCV), or exposure to drugs such as aflatoxin B1. including breast and lung cancer (20). E-cadherin is intimately Many more studies have shown that multiple genetic related to malignancy which is characterized by uncontrolled alterations are associated with HCC, but the molecular proliferation, dedifferentiation, invasion and metastasis, and mechanisms of hepatocarcinogenesis are not yet clear. its expression is reduced by hypermethylation of the promoter region in a number of tumors (17, 21). Furthermore, a cluster of cadherin genes are located on chromosome 16q, which is commonly deleted in various types of human cancer including Correspondence to: Suguru Yamada, Department of Surgery II, HCC, with deletion rates ranging from 28% to 70% (22, 23). Graduate School and Faculty of Medicine, University of Nagoya, Therefore, it has been proposed that genetic and epigenetic 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel: +8152 7442249, Fax: +81527442255, e-mail: [email protected] alterations in CDH might be involved in liver carcinogenesis (24-26). Key Words: Hepatocellular carcinoma, cadherin, methylation, In this study, we examined the methylation status of CDH prognosis. genes, located at 16q, using a methylation-specific polymerase

0250-7005/2007 $2.00+.40 2269 ANTICANCER RESEARCH 27: 2269-2274 (2007) chain reaction (PCR), and evaluated the correlation with Table I. Methylation stutus and mRNA expression of M-cadherin in HCC. clinicopathological parameters. M-cadherin M-cadherin

Materials and Methods Case Methylation RT-PCR Case Methylation RT-PCR status status Patients and specimens. Cancerous tissues and the surrounding non- cancerous hepatic parenchyma were obtained from 47 primary 1-↓ 25 - ↓ HCC patients who underwent resection at Nagoya University 2+↓ 26 + ↑ Hospital, Japan, during the period from May 1994 to December 3+↓ 27 - ↑ 1997. The study was approved by the Ethics Committee of the 4+↑ 28 + → hospital. Informed consent was obtained from all patients for the 5-↑ 29 + ↓ ↑ ↑ subsequent use of their resected tissues. There were 43 male and 4 6- 30 + → ↓ female patients, with ages ranging from 21 to 77 years (mean, 61 7+ 31 - ↓ → years). The mean follow-up period in the prognosis study was 8- 32 + → ↓ 64.3±31.9 (mean±SD) months. Tumor sizes ranged from 1.2 cm 9+ 33 - 10 - ↓ 34 - ↑ to 14.0 cm, with a mean size of 3.9±2.7 cm. Specimens were 11 + ↓ 35 + ↑ classified histologically using the Japanese staging system of the 12 + ↓ 36 + ↑ Liver Cancer Study Group of Japan (27). 13 + ↑ 37 - ↑ 14 + ↑ 38 - ↑ Bisulfite modification and methylation-specific polymerase chain 15 + ↓ 39 - ↓ reaction (MSP). DNA from primary HCC tissues and 16 + ↓ 40 + ↓ corresponding non-cancerous tissues were subjected to bisulfite 17 + → 41 - ↑ treatment. Briefly, 2 Ìg of DNA were denatured with NaOH and 18 - → 42 + ↓ modified with sodium bisulfite. DNA samples were then purified 19 - ↓ 43 - ↓ using the Wizard purification resin (Promega Corp., Madison, WI, 20 - → 44 + → USA), treated again with NaOH, precipitated with ethanol, and 21 + ↓ 45 - ↑ resuspended in water. The modified DNA was used as a template 22 + ↓ 46 + → for MSP. The PCR amplification of modified DNA samples 23 + → 47 - → consisted of 35 cycles of 94ÆC for 15 s, 60ÆC for 12 s and 72ÆC for 24 - ↑ 10 s, after the initial denaturation step (94ÆC for 5 min). Abbreviations: +: methylated; -: unmethylated; ↑: higher than normal → ↓ RT-PCR. The expression of M-cadherin mRNA was analyzed using liver; : same as normal liver; : lower than normal liver. RT-PCR. Total RNA (10 Ìg) isolated from primary HCC tissues and corresponding non-cancerous tissues were used to generate complementary DNA (cDNA) and then amplified by PCR primers for M-cadherin: 5’-TGACTACGAGGGTGACGGCT-3’(forward) methylation was detected at a ratio ranging from 0% to and 5’-GGAGTGGCATGTCCAGTTGC-3’(reverse), which 55.3% (Figure 1A). In particular, M-cadherin (CDH15) was amplify a 263 bp product. The PCR amplification consisted of 30 the most hypermethylated of all 7 CDH genes. cycles of 94ÆC for 15 s, 60ÆC for 15 s and 72ÆC for 12 s, after the Representative cases of HCC tissues are shown in Figure 1B. initial denaturation step (94ÆC for 5 min). Expression of M-cadherin mRNA in HCC. We examined the Statistical analysis. To analyze the correlation between M-cadherin methylation status and clinicopathological parameters, differences expression of M-cadherin mRNA using RT-PCR in primary in the numerical data between the two groups were evaluated HCC tissues and corresponding non-cancerous tissues. In using Fisher’s exact test or ¯2 test. Overall survival rates were 20 (42.5%) of 47 cases, the M-cadherin expression of calculated using the Kaplan-Meier method, and the difference in primary HCC tissues was less than that of corresponding survival curves was analyzed using the log-rank test. Independent non-cancerous tissues (Table I). Representative cases are prognostic factors were analyzed with the Cox proportional shown in Figure 1C. In 20 cases with lower expression of hazards regression model in a stepwise manner. Data are M-cadherin mRNA, 11 cases (55%) were hypermethylated expressed as mean±SD. The presence of a statistically significant difference was denoted by p<0.05. (Table I).

Results Correlation between M-cadherin methylation status and clinicopathological parameters in HCC. The association Hypermethylation of the CDH promoter region in HCC. To between M-cadherin methylation status and investigate hypermethylation of the CDH promoter region, clinicopathological parameters in patients with HCC was MSP was performed in 47 primary HCC tissues and analyzed using Fisher’s exact test or ¯2 test statistically corresponding non-cancerous tissues. We examined 7 CDH (Table II). M-cadherin methylation was seen in well- genes on chromosome 16q. In primary tissues, hyper- differentiated cancers, and more frequently in larger

2270 Yamada et al: Frequent Promoter Methylation of M-cadherin in HCC

Figure 1. A) Hypermethylation of CDH genes, located on 16q, in HCC. N: non-cancerous tissue, T: HCC tissue, U: Unmethylated, M: Methylated. B) Promoter hypermethylation of M-cadherin in HCC tissues. The representative cases are shown. C) The expression of M-cadherin mRNA in HCC tissues using RT-PCR.

tumors, although the differences were not statistically vascular invasion, liver cirrhosis and pathological TNM significant. No significant association was found between stage), and it was revealed that M-cadherin methylation M-cadherin methylation status and other variables. status and liver cirrhosis remained the strongest variables for independently predicting overall survival (p=0.011 and Univariate and multivariate analyses of prognostic factors for p=0.024, respectively; Table III). patients with HCC. We analyzed the overall survival rates to assess the prognostic significance of M-cadherin Discussion methylation status. The five-year overall survival rate of the 47 patients with HCC was 74.1%. Patients with methylated Cadherins form a cluster in this chromosomal region and M-cadherin had shorter 5-year survival rates than patients are also considered prime candidates for tumor suppressor with unmethylated M-cadherin (overall survival rate, 67.4% genes. M-cadherin, a calcium-dependent intercellular vs. 82.7%; p=0.0167; Figure 2) when assessed using adhesion molecule, is expressed in skeletal muscle cells. It Kaplan-Meier curves. is important for skeletal muscle development, in particular To evaluate the potential role of M-cadherin methylation the fusion of myoblasts into myotubes (28). E-cadherin status in determining the postoperative prognosis of HCC mediates cell-cell interaction in a calcium-dependent patients, univariate analysis was conducted. The results manner. The extracellular domain of E-cadherin is showed that M-cadherin methylation was a significant important for cell-cell interaction, and the intracellular predictor of overall survival (p=0.0215). Moreover, domain binds to catenins that attach to the actin multivariate analysis was performed with 6 other prime cytoskeleton (9). In tumor model systems, down-regulation variables (·-fetoprotein, tumor multiplicity, tumor size, of E-cadherin has been associated with loss of intercellular

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Table II. The correlation between M-cadherin methylation status and Table III. Multivariate analysis for overall survival of patients with HCC. clinicopatholagical parameters in HCC. Overall survival Clinicopathological Number M-cadherin P-value parameters of cases methylation 95% (number) Variable Odds Confidence P-value ratio interval Methylated Un- methylated M-cadherin methylation status Age (present: absent) 4.47 1.40-14.2 0.011 <60 13 6 7 0.435 AFP (≥20 ng/ml: <20 ng/ml) 1.45 0.56-3.76 0.447 ≥60 34 20 14 Tumor multiplicity (multiple: solitary) 0.99 0.27-3.58 0.985 Gender Tumor size (≥3.5 cm: <3.5 cm) 0.47 0.11-1.94 0.298 Male 43 25 18 0.202 Vascular invasion (present: absent) 2.24 0.37-13.7 0.382 Female 4 1 3 Liver cirrhosis (present: absent) 3.28 1.17-9.18 0.024 Virus Pathological stage (I and II: III and IV) 1.45 0.17-12.5 0.736 HBV 6 2 4 0.478 HCV 37 22 15 None 4 2 2 Pugh-Child's classification A 43 23 20 0.408 B431 AFP adhesiveness, which may result in initiation of invasion, <20 ng/ml 17 9 8 0.883 tumor dedifferentiation and the destruction of normal ≥20 ng/ml 29 16 13 PIVKA II tissue morphology (17, 21). Other cadherin family genes <0.1 AU/ml 23 13 10 0.847 are localized in this region and could also be involved in ≥0.1 AU/ml 15 8 7 the tumorigenesis of liver cells. The present study provides Tumor multiplicity the first evidence of reduced M-cadherin expression in Solitary 32 17 15 0.659 Multiple 15 9 6 HCC tissues, with the reduction corresponding to the Histologic type of tumor reduced survival of HCC patients. Well-differentiated 5 5 0 0.062 Because hypermethylation of the E-cadherin promoter Moderately-differentiated 41 20 21 Poorly-differentiated 1 0 1 has been observed at significant rates in HCC (25, 26), Tumor size we examined the methylation status of CDH genes on <3.5cm 29 13 16 0.063 chromosome 16q using MSP. In primary tissues, M- ≥3.5cm 18 13 5 cadherin was the most hypermethylated of 7 CDH genes. Pattern of tumor growth Expansive 40 22 18 0.832 Clinically, those patients with methylated M-cadherin Infiltrative 5 3 2 displayed a significantly poorer prognosis. Because M- Formation of fibrous capsule cadherin is mainly expressed in skeletal muscle cells and Present 38 21 17 0.707 Absent 8 5 3 there was no association between methylation status and Capsular infiltration clinico-pathological parameters, it is difficult to explain Present 36 20 16 >0.999 this result. However, the cadherins form a cluster in this Absent 12 5 7 chromosomal region, in particular, M-cadherin is found Septal formation Present 37 19 18 0.222 near E-cadherin, and we suspect that this fact could Absent 8 6 2 explain the result. Further study of the mechanisms Vascular invasion between cadherins and functional research of M-cadherin Present 10 6 4 0.737 Absent 37 20 17 is needed. Liver cirrhosis Present 17 8 9 0.391 Conclusion Absent 30 18 12 Surgical margin Negative 24 15 9 0.316 M-cadherin is hypermethylated in HCC and is Positive 21 10 11 significantly associated with overall survival. Detection of Pathological Stage M-cadherin hypermethylation in surgically excised HCC I 5 3 2 0.989 II 28 15 13 tissues may provide a new strategy for cancer prevention, III 9 5 4 an adjuvant therapy, and a potential biomarker for the IVA 5 3 2 prognostication of HCC. Further investigation is needed to understand how M-cadherin methylation contributes to HBV: hepatitis B virus; HCV: hepatitis C virus; AFP: alpha-fetoprotein; PIVKAII: induced by vitamin K absence or antagonists-II. the pathogenesis of HCC.

2272 Yamada et al: Frequent Promoter Methylation of M-cadherin in HCC

Figure 2. Patients with methylated M-cadherin had shorter 5-year survival rates than patients with unmethylated M-cadherin (overall survival rate, 67.4% vs. 82.7%; p=0.0167) when assessed using Kaplan-Meier curves.

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